One of the hallmarks of Alzheimer's disease (AD) is neurofibrillary degeneration which results from the aggregation of phosphorylated tau proteins into paired helical filament (PHF) structures. AD2 is a new monoclonal antibody raised against PHF tau which detects neurofibrillary tangles in AD brain. In primary neuronal cultures, phorbol ester treatment induced a time- and dose-dependent increase in AD2 immunoreactivity quantified by laser confocal microscopy and immunoblottings. Alkaline phosphatase treatment reversed these immunocytochemical changes. These results suggest that the modifications of neuronal metabolism induced by phorbol ester including protein kinase C activation produce an increase in phosphorylated tau immunoreactivity.

Alzheimer's disease is characterized by an intraneuronal aggregation of hyperphosphorylated tau proteins into paired helical filaments. The hyperphosphorylation of tau proteins induces a decrease in their electrophoretic mobility, resulting in a pathological tau triplet referred to as tau 55, 64 and 69 or tau-PHF. We have developed monoclonal antibodies directed against this pathological tau triplet. In the present article, we report the properties of antibody AD2, which detects the hyperphosphorylated tau proteins forming paired helical filaments during Alzheimer's disease. Using immunoblotting, AD2 exclusively labeled the tau triplet, while normal tau proteins from control cases were not immunodetected. Furthermore, AD2 is highly specific in that it was able to detect the triplet not only in tau preparations but also in total brain homogenates from Alzheimer's disease patients. The binding of this monoclonal antibody to tau proteins is phosphorylation dependent. Characterization of this antibody allowed us to identify its epitope as containing phosphorylated Ser-396 with the participation of phosphorylated Ser-404. AD2 was also shown to label normal tau proteins from rapidly processed brain tissues, but its epitope is rapidly dephosphorylated during postmortem intervals. However, in autopsic brains, AD2 still represents a valuable tool to investigate neurofibrillary degeneration at the biochemical and immunocytochemical levels.

Tau in PHF is known to be highly phosphorylated and immunochemical study has indicated the similarity of the phosphorylation between PHF-tau and fetal tau. We have determined the exact phosphorylation sites in both PHF-tau and fetal rat tau by ion-spray mass spectrometry and sequencing of ethanethiol-modified peptides. In PHF-tau, 19 sites have been identified; all the phosphorylation sites except for Ser-262 are localized to the amino- and carboxyl-terminal flanking regions of the microtubule-binding domain. Half of them are shared by fetal tau. Thus, PHF-tau is much more phosphorylated. Whereas most of the sites in fetal tau are proline-directed, half of them in PHF-tau are nonproline-directed. Overall, the hyperphosphorylation of PHF-tau can be considered to consist of fetal-type phosphorylation and additional proline-directed and nonproline-directed phosphorylation. This extraphosphorylation may provide PHF-tau with the unusual characteristics including assembly incompetence.

Monoclonal antibodies against human paired helical filament tau (PHF-tau) proteins were produced. Two of these antibodies, AD1 and AD2, were shown by immunoblot to be directed against distinct hyperphosphorylated epitopes of the PHF-tau proteins. Using AD1 and AD2, an antigen-capture ELISA specific for PHF-tau proteins was developed and used to map the neurofibrillary degeneration of several Broadmann areas from an Alzheimer's disease patient. The results confirm that the neurofibrillary degeneration predominates in parietal and temporal lobes.

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.

Two monoclonal antibodies (mAbs), 10-2 and 10-5, both directed against recombinant hirudin variant 2-Lys47 (rHV2), were selected for their high affinity and epitopic specificities to develop a two-site immunoassay of rHV2. The mAb concentrations, incubation time, and temperature were optimized. The immunoassay has a detection limit for rHV2 of 45 ng/L in plasma and 30 ng/L in urine. The reactivity of the mAbs was tested against rHV2 and several forms of this protein truncated in the carboxyl terminus. The capture mAb 10-2 was found to be mainly directed against rHV2, whereas tracer mAb 10-5 was independent of the carboxyl-terminal region of the protein. This explains the high specificity of the immunoassay for the 65-amino acid form of hirudin.